parallel computing

Loading Data

To load 10x data there a several options with pros an cons.

## [1] "RERUNstate= FALSE"

batch info

Samples used in this project
SampleID Samplename Repl group cells_seq
2_C 2C R_2 C 5253
2_L 2L R_2 L 2708
2_S 2S R_2 S 3879
235_d12 235d12 R_235 d12 3066
3_C 3C R_3 C 4903
3_L 3L R_3 L 2580
3_S 3S R_3 S 4650
4_C 4C R_4 C 4948
4_L 4L R_4 L 1860
4_S 4S R_4 S 3610

Pre-processing and QC

##                       orig.ident nCount_RNA nFeature_RNA percent.mt percent.rb
## 2C_AAACCTGAGTCGAGTG-1         2C       7717         2210  0.5572114   20.29286
## 2C_AAACCTGCAGCCAGAA-1         2C       5075         1833  0.3349754   17.87192
## 2C_AAACCTGCAGGGCATA-1         2C       5507         1814  0.8534592   24.62321
## 2C_AAACCTGCATTTCAGG-1         2C       8181         2509  0.7089598   17.69955
## 2C_AAACCTGGTCGCTTCT-1         2C       3193         1216  0.7203257   16.72408
##                       keepall keepnCRlow keepM keepR pANN_0.25_0.09_432
## 2C_AAACCTGAGTCGAGTG-1    TRUE       TRUE  TRUE  TRUE          0.2785924
## 2C_AAACCTGCAGCCAGAA-1    TRUE       TRUE  TRUE  TRUE          0.3724340
## 2C_AAACCTGCAGGGCATA-1    TRUE       TRUE  TRUE  TRUE          0.1260997
## 2C_AAACCTGCATTTCAGG-1    TRUE       TRUE  TRUE  TRUE          0.3255132
## 2C_AAACCTGGTCGCTTCT-1    TRUE       TRUE  TRUE  TRUE          0.2756598
##                       DF.classifications_0.25_0.09_432 pANN_0.25_0.09_176
## 2C_AAACCTGAGTCGAGTG-1                          Singlet                 NA
## 2C_AAACCTGCAGCCAGAA-1                          Singlet                 NA
## 2C_AAACCTGCAGGGCATA-1                          Singlet                 NA
## 2C_AAACCTGCATTTCAGG-1                          Singlet                 NA
## 2C_AAACCTGGTCGCTTCT-1                          Singlet                 NA
##                       DF.classifications_0.25_0.09_176 pANN_0.25_0.09_319
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_319 pANN_0.25_0.09_252
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_252 pANN_0.25_0.09_403
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_403 pANN_0.25_0.09_168
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_168 pANN_0.25_0.09_382
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_382 pANN_0.25_0.09_407
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_407 pANN_0.25_0.09_106
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_106 pANN_0.25_0.09_297
## 2C_AAACCTGAGTCGAGTG-1                             <NA>                 NA
## 2C_AAACCTGCAGCCAGAA-1                             <NA>                 NA
## 2C_AAACCTGCAGGGCATA-1                             <NA>                 NA
## 2C_AAACCTGCATTTCAGG-1                             <NA>                 NA
## 2C_AAACCTGGTCGCTTCT-1                             <NA>                 NA
##                       DF.classifications_0.25_0.09_297 SampleID Repl group
## 2C_AAACCTGAGTCGAGTG-1                             <NA>      2_C  R_2     C
## 2C_AAACCTGCAGCCAGAA-1                             <NA>      2_C  R_2     C
## 2C_AAACCTGCAGGGCATA-1                             <NA>      2_C  R_2     C
## 2C_AAACCTGCATTTCAGG-1                             <NA>      2_C  R_2     C
## 2C_AAACCTGGTCGCTTCT-1                             <NA>      2_C  R_2     C

## [1] "Scatter nCounts vs n Features"

Filtering

Normalizing and var Features

##  [1] "Gzma"     "Ccl4"     "Ifitm1"   "Trbv17"   "Ccl1"     "Penk"    
##  [7] "Hbb-bs"   "Hba-a1"   "Ecm1"     "Areg"     "Cxcl2"    "Gcg"     
## [13] "Hba-a2"   "Cxcl10"   "Trbv1"    "Ccl3"     "Trbv15"   "Hbb-bt"  
## [19] "Trbv29"   "Trav8d-2"

Split object in batchs

Intergate function of Seurat leads to lost processes on the cluster, forking problem.

Scaling of the data

Perform integration

Perform integrated analysis

PCA

## PC_ 1 
## Positive:  Vps37b, Bhlhe40, Ifrd1, Tnfaip3, Hilpda 
## Negative:  Rps8, Rps20, Rpl28, Rpl10a, Ifi47 
## PC_ 2 
## Positive:  AW112010, Ifi27l2a, Ly6a, S100a11, Cxcr3 
## Negative:  Rpl13, Rps2, Rpl32, Rps5, Igfbp4 
## PC_ 3 
## Positive:  Birc5, Stmn1, Pclaf, Ccna2, Spc24 
## Negative:  Rpl13, Rps4x, Rps5, Rps18, Rpl8 
## PC_ 4 
## Positive:  S100a6, Vim, Lgals3, Nkg7, Crip1 
## Negative:  Izumo1r, Stat1, Cd74, Cd81, Cd83 
## PC_ 5 
## Positive:  Lgals1, Vim, Actg1, S100a10, S100a6 
## Negative:  Zfp36l2, Jun, Evl, Cd7, Btg2
VizDimLoadings(SObj, dims = 1:2, reduction = "pca")

DimPlot(SObj, reduction = "pca",label=TRUE,group.by="orig.ident")

DimPlot(SObj,reduction="pca",split.by="group",group.by="orig.ident",label=TRUE)+
    geom_hline(yintercept=0)+geom_vline(xintercept=0)

DimPlot(SObj, reduction = "pca",label=TRUE,group.by="orig.ident",dims=c(2,3))

DimPlot(SObj,reduction="pca",dims=c(2,3),split.by="group",group.by="orig.ident",label=TRUE)

Heatmaps to decide which PCs to include, find batch effects

DimHeatmap(SObj, dims = 1, cells = 500, balanced = TRUE)

DimHeatmap(SObj, dims = 1:17, cells = 500, balanced = TRUE)

## Inspect data for SCT

##Determine Dimensionality of Data set:

Cluster cells

## 2C_AAACCTGAGTCGAGTG-1 2C_AAACCTGCAGCCAGAA-1 2C_AAACCTGCAGGGCATA-1 
##                     0                     0                     0 
## 2C_AAACCTGCATTTCAGG-1 2C_AAACCTGGTCGCTTCT-1 
##                     0                     0 
## Levels: 0 1 2 3 4 5 6 7 8 9 10 11 12

UMAP

Vis Treg Genes

Find Diff features

## 
## 
## |        | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-------|-----:|----------:|-----:|-----:|---------:|
## |Neurl3  |     0|  1.0663105| 0.681| 0.250|         0|
## |Il18rap |     0|  0.4520373| 0.281| 0.101|         0|
## |Stat4   |     0|  1.1966809| 0.835| 0.472|         0|
## |Stk17b  |     0|  0.6193758| 0.960| 0.849|         0|
## |Coq10b  |     0|  0.5059204| 0.502| 0.264|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Ptpn18 |     0|  0.4603764| 0.986| 0.979|         0|
## |Neurl3 |     0| -0.8881515| 0.142| 0.389|         0|
## |Ctla4  |     0| -1.6912062| 0.603| 0.843|         0|
## |Cd48   |     0|  0.6016163| 0.843| 0.742|         0|
## |Dusp10 |     0| -0.9277337| 0.058| 0.288|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Neurl3 |     0|  0.8736130| 0.592| 0.296|         0|
## |Arid5a |     0|  0.8095186| 0.527| 0.342|         0|
## |Stk17b |     0|  0.8925751| 0.954| 0.857|         0|
## |Coq10b |     0|  0.7786504| 0.520| 0.276|         0|
## |Clk1   |     0|  0.8524342| 0.676| 0.404|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Ctla4  |     0| -1.9626862| 0.578| 0.825|         0|
## |Icos   |     0| -1.1836658| 0.638| 0.815|         0|
## |Rgs1   |     0| -1.8963865| 0.508| 0.751|         0|
## |Slamf6 |     0|  0.7655931| 0.533| 0.252|         0|
## |Ephx1  |     0|  0.8856907| 0.469| 0.140|         0|
## 
## 
## |        | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-------|-----:|----------:|-----:|-----:|---------:|
## |Zap70   |     0|  0.6929217| 0.891| 0.758|         0|
## |Cst7    |     0|  1.0398208| 0.924| 0.700|         0|
## |S100a11 |     0|  0.5707854| 0.990| 0.918|         0|
## |Ctss    |     0|  0.7796210| 0.760| 0.497|         0|
## |Vps37b  |     0| -2.4479675| 0.164| 0.552|         0|
## 
## 
## |        | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-------|-----:|----------:|-----:|-----:|---------:|
## |Ramp1   |     0|  1.0998419| 0.457| 0.164|         0|
## |Vim     |     0|  1.0934467| 0.994| 0.879|         0|
## |Cdc25b  |     0|  0.9080684| 0.568| 0.204|         0|
## |Rps27   |     0| -0.4276138| 0.997| 0.997|         0|
## |S100a13 |     0|  0.6007740| 0.974| 0.853|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Rpl7   |     0|  0.8389506| 0.997| 0.983|         0|
## |Zap70  |     0| -1.1414245| 0.610| 0.783|         0|
## |Stat1  |     0| -1.4139616| 0.540| 0.780|         0|
## |Dnah7a |     0|  0.7226934| 0.280| 0.024|         0|
## |Stk17b |     0| -1.1913404| 0.799| 0.880|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Stat1  |     0|   1.557797| 0.988| 0.756|         0|
## |Ifi214 |     0|   1.376515| 0.699| 0.198|         0|
## |Ifi209 |     0|   1.800837| 0.956| 0.493|         0|
## |Ifi208 |     0|   1.865837| 0.866| 0.301|         0|
## |Ifih1  |     0|   1.319685| 0.604| 0.152|         0|
## 
## 
## |        | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-------|-----:|----------:|-----:|-----:|---------:|
## |Ptma    |     0|  1.3117789| 1.000| 0.996|         0|
## |Cenpf   |     0|  1.2002792| 0.414| 0.013|         0|
## |Spc25   |     0|  0.5792946| 0.284| 0.015|         0|
## |Selenoh |     0|  1.6368082| 0.896| 0.327|         0|
## |Ccdc34  |     0|  0.6898874| 0.421| 0.059|         0|
## 
## 
## |        | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-------|-----:|----------:|-----:|-----:|---------:|
## |Slc40a1 |     0|   2.344474| 0.519| 0.003|         0|
## |Fcer1g  |     0|   3.387036| 0.751| 0.025|         0|
## |Gatm    |     0|   1.489229| 0.388| 0.004|         0|
## |Slpi    |     0|   2.057951| 0.373| 0.008|         0|
## |Vcam1   |     0|   1.997361| 0.435| 0.002|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Cxcl10 |     0|   3.725377| 0.624| 0.066|         0|
## |Rsad2  |     0|   2.257266| 0.668| 0.056|         0|
## |Ifit3  |     0|   2.828317| 0.809| 0.104|         0|
## |Ifit3b |     0|   1.671574| 0.470| 0.041|         0|
## |Ifit1  |     0|   2.945491| 0.881| 0.185|         0|
## 
## 
## |       | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:------|-----:|----------:|-----:|-----:|---------:|
## |Knl1   |     0|  0.7875364| 0.373| 0.017|         0|
## |Rad51  |     0|  0.9049908| 0.546| 0.021|         0|
## |Nusap1 |     0|  0.9009919| 0.394| 0.021|         0|
## |Tpx2   |     0|  1.0250170| 0.470| 0.030|         0|
## |Cks1b  |     0|  1.2148313| 0.679| 0.065|         0|
## 
## 
## |      | p_val| avg_log2FC| pct.1| pct.2| p_val_adj|
## |:-----|-----:|----------:|-----:|-----:|---------:|
## |Lmo2  |     0|   2.176814| 0.598| 0.003|         0|
## |Gatm  |     0|   1.455458| 0.392| 0.007|         0|
## |Car1  |     0|   3.773360| 0.716| 0.009|         0|
## |Gstm5 |     0|   1.700562| 0.480| 0.008|         0|
## |Rhd   |     0|   2.638066| 0.402| 0.001|         0|
## Calculating cluster 0
## Calculating cluster 1
## Calculating cluster 2
## Calculating cluster 3
## Calculating cluster 4
## Calculating cluster 5
## Calculating cluster 6
## Calculating cluster 7
## Calculating cluster 8
## Calculating cluster 9
## Calculating cluster 10
## Calculating cluster 11
## Calculating cluster 12
## [1] "gene list ordered by avg_log2FC"

## Selecting by gene
## [1] "gene list ranked by seurat"

TCR visualisation

## [1] "TCRs included: 55\nTCRs leftout: 28"

## [1] "TCRs included: 57\nTCRs leftout: 39"

## [1] "TCRs included: 60\nTCRs leftout: 30"

Session Info

## R version 4.0.3 (2020-10-10)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Debian GNU/Linux 10 (buster)
## 
## Matrix products: default
## BLAS/LAPACK: /misc/software/package/RBioC/libmkl/R-4.0.3_buster_intel-mkl-64bit-2020.1-102_build/compilers_and_libraries_2020.1.217/linux/mkl/lib/intel64_lin/libmkl_gf_lp64.so
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] dplyr_1.0.3        patchwork_1.1.1    ggplot2_3.3.3      cowplot_1.1.1     
## [5] future_1.21.0      knitr_1.30         SeuratObject_4.0.0 Seurat_4.0.0      
## [9] rmarkdown_2.6     
## 
## loaded via a namespace (and not attached):
##   [1] nlme_3.1-149         matrixStats_0.57.0   RcppAnnoy_0.0.18    
##   [4] RColorBrewer_1.1-2   httr_1.4.2           sctransform_0.3.2   
##   [7] tools_4.0.3          R6_2.5.0             irlba_2.3.3         
##  [10] rpart_4.1-15         KernSmooth_2.23-17   uwot_0.1.10         
##  [13] mgcv_1.8-33          DBI_1.1.1            lazyeval_0.2.2      
##  [16] colorspace_2.0-0     withr_2.4.1          tidyselect_1.1.0    
##  [19] gridExtra_2.3        compiler_4.0.3       plotly_4.9.3        
##  [22] labeling_0.4.2       scales_1.1.1         lmtest_0.9-38       
##  [25] spatstat.data_1.7-0  ggridges_0.5.3       pbapply_1.4-3       
##  [28] goftest_1.2-2        spatstat_1.64-1      stringr_1.4.0       
##  [31] digest_0.6.27        spatstat.utils_2.0-0 pkgconfig_2.0.3     
##  [34] htmltools_0.5.1.1    parallelly_1.23.0    limma_3.46.0        
##  [37] highr_0.8            fastmap_1.1.0        htmlwidgets_1.5.3   
##  [40] rlang_0.4.10         shiny_1.6.0          farver_2.0.3        
##  [43] generics_0.1.0       zoo_1.8-8            jsonlite_1.7.2      
##  [46] ica_1.0-2            magrittr_2.0.1       Matrix_1.2-18       
##  [49] Rcpp_1.0.6           munsell_0.5.0        abind_1.4-5         
##  [52] reticulate_1.18      lifecycle_0.2.0      stringi_1.5.3       
##  [55] yaml_2.2.1           MASS_7.3-53          Rtsne_0.15          
##  [58] plyr_1.8.6           grid_4.0.3           parallel_4.0.3      
##  [61] listenv_0.8.0        promises_1.1.1       ggrepel_0.9.1       
##  [64] crayon_1.3.4         deldir_0.2-9         miniUI_0.1.1.1      
##  [67] lattice_0.20-41      splines_4.0.3        tensor_1.5          
##  [70] pillar_1.4.7         igraph_1.2.6         future.apply_1.7.0  
##  [73] reshape2_1.4.4       codetools_0.2-16     leiden_0.3.7        
##  [76] glue_1.4.2           evaluate_0.14        data.table_1.13.6   
##  [79] vctrs_0.3.6          png_0.1-7            httpuv_1.5.5        
##  [82] polyclip_1.10-0      gtable_0.3.0         RANN_2.6.1          
##  [85] purrr_0.3.4          tidyr_1.1.2          scattermore_0.7     
##  [88] assertthat_0.2.1     xfun_0.20            mime_0.9            
##  [91] xtable_1.8-4         later_1.1.0.1        survival_3.2-7      
##  [94] viridisLite_0.3.0    tibble_3.0.5         cluster_2.1.0       
##  [97] globals_0.14.0       fitdistrplus_1.1-3   ellipsis_0.3.1      
## [100] ROCR_1.0-11